VIRUS DU PAPILLOME HUMAIN, TYPE 57; SEQUENCE DE CE VIRUS ET PROTEINES CODEES PAR CETTE SEQUENCE

HUMAN PAPILLOMA VIRUS TYPE 57, THE DNA THEREOF AND THE PROTEINS ENCODED THEREBY

Abrégé anglais

The invention describes the isolation for the first time
of human papilloma virus (HPV) 57, the partial
characterization of its genome and its cloning in pUC 19. This
opens up a way of diagnosing those tumors (oral, genital
and cutaneous tumors) which are associated with HPV 57.

Revendications

- 5 -
THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. Isolated HPV 57 DNA characterized by the restriction
pattern as shown in Figure 2.
2. HPV 57 DNA in pUC 19 deposited and defined as DSM 4694.
3. Isolated human papilloma virus (HPV) 57 including
variants and mutants characterized by the HPV 57 DNA as
claimed in claim 1 or 2.
4. The use of the DNA as claimed in claim 1 or 2 for the
preparation of proteins by genetic manipulation.
5. A method for the diagnosis of HPV 57 infections, which
comprises hybridizing the RNA or DNA which is to be examined
with DNA as claimed in claim 1 or 2.
6. A diagnostic aid containing DNA as claimed in claim 1 or
2.
7. The use of HPV 57 as claimed in claim 3 for the
preparation of a vaccine.
8. The use of the DNA as claimed in claim 4 for the
preparation of a vaccine.

Description

1339268
BEHRIN~w~ ARTIENGESELLSCHAFT
Human Pspilloma virus Type 57, the DNA thereof and the
proteins encoded thereby
The human papilloma viruses (HPV) form a group of more
than 50 different types. HPV has been found sssociated
with benign (warts, condylomas in the genital region) and
malignant (carcinomas of the skin and the vagina) epithe-
lial neoplasms. Papilloma viruses cannot be grown inculture. Thus, methods of genetic manipulation are
required for the use of human papilloma virus type 57 DNA
(HPV 57 DNA) as a diagnostic aid and for obtA~ning the
expression product~, for using them as antigens, for
isolating antibodies and for preparing corresponding
diagnostic aids and therapeutic agent~.
The invention is based on the isolation for the first
time of HPV 57, partial characterization of its genome
and cloning in pUC 19. This opens up a way of diagnosing
tumors (oral, genital and cutaneous tumor~) as~ociated
with HPV 57.
The invention will now be described in relation to the
drawings, in which:
Figure 1 shows the colinearity of HPV 57 with HPV 18
based on hybridization experiments; and
Figure 2 shows physical genome maps of HPV 57.
The invention i8 defined in the patent claims. Further
embodiments of the invention are described in detail
hereinafter.
The cloning of HPV 57 made it possible to compare with 56
other HPVs. HPV 2 and HPV 27 are very closely related,
and hybridization in liquid phase revealed 17% and 25%
homology, respectively. The colinearity of HPV 57 with
HPV 2 and 27 was determined (Fig. 1) and a physical
genome map for restriction enzyme cleavages (Fig. 2) was
constructed.

- 2 - 1339268
This has opened up a way of testing neoplasms (genital
and cutaneous tumors), especially tumors on the head, for
the presence of HPV 57 and, where appropriate, a thera-
peutic approach via antibodies to HPV 57 proteins.
Examples
1. Isolation of episomal HPV 57 DNA
High molecular weight DNA was isolated from an
inverted papilloma of the maxillary sinus as descri-
bed (Gi6smann et al. (1982), Int. J. Cancer 29, 143
- 146).
2. Cloning of HPV 57 in the plasmid pUC 19
The known plasmid pUC 19 (Yani~h-Perron et al.
(1985), Gene 33, 113 - 119) was chosen as cloning
vector. HPV 57-contAin~ng cellular double-stranded
DNA was cleaved with Eco RI and then cloned into p~C
19 (MAni~tis et al. (1982) Molecular Cloning: A
Laboratory MAnllAl; Cold Spring Harbor Laboratory
Press, New York). Recombinant clones were identified
in the ~-galactosidase test (Messing et al. (1977),
Proc. Nat. Acad. Sci. USA 75, 3641 - 3646) and rapid
DNA extraction was followed by analysis of DNA
fragments ligated in (Birnboim, H.L. and Doly, J.
(1979), Nucl. Acids. Res. 7, 1513 - 1523).
3. Physical genome maps of HPV 57
HPV 57 DNA detached from the vector by Eco RI
cleavage was digested with re~triction endonucleases
and the corresponding physical genome maps were
constructed by generally known methods. The result
i8 summarized in Fig. 2, the sole Eco RI cleavage
site being used to linearize the HPV 57 molecule.
The table shows the length of the individual res-
triction fragments.

4. Comparison with other HPVs 133 9 2 68
The DNA of the HPV 57 genome was compared by means
of DNA/DNA hybridization under various degrees of
stringency with the DNAs of 56 available HPV types
(E.M. Southern (1975), J. Mol. Biol. 98, 50-517).
Under high-stringency conditions (melting tempera-
ture Tm -20~C) HPV 57 DNA hybridizes with HPV 2 and
HPV 27.
The colinearity with HPV 18 based on hybridization
experiments is shown in Fig. 1.
Rnowing the HPV 18 DNA ~equence (Cole et al. (1987) J.
Mol. Biol. 193, 599 - 608) it is possible to deduce the
open reading frames of HPV 57 and thus to obtain the HPV
57 proteins by general methods for subcloning with
subsequent expression in prokaryotic or eukaryotic
expression systems.
The pUC 19 plasmid DNA contAining HPV 57 was deposited on
June 13, 1988, at the Deutsche Sammlung fur Mikroorganis-
men (German Microorganism Collection) under the number
DSM 4694 in accordance with the Budapest Treaty.
Rey to Fig.2
The circular genome was cloned via its Eco RI clearage
site, the figures following the letters designating the
fragments indicate the length in base-pairs (bp). Restri-
ction enzymes with one cleavage site are XhoI, HindII andSacI. The restriction enzymes XbaI, SalI, BamHI, HindIII
and HpaI do not cleave. The exact location of the four
500 bp-long PstI fragments was not determined.

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